Effects of lyophilization on human amniotic membrane.
OBJECTIVE
This study aimed to evaluate the effects of lyophilization and cryopreservation on human amniotic membrane (HAM) in terms of histological characteristics and growth factor levels.


METHODS
Non-preserved, lyophilized and cryopreserved HAM samples from 13 placentas were investigated. The morphological characteristics of HAM were evaluated using light and electron microscopy. Immunohistochemical methods were also applied to assess the distribution of collagen IV in the basement membrane. Total protein amounts were measured in extracts of intact amniotic membrane from non-preserved, lyophilized and cryopreserved samples. An enzyme-linked immunosorbent assay (ELISA) was used to assay growth factor protein levels for epidermal growth factor, fibroblast growth factor basic, hepatocyte growth factor, keratinocyte growth factor, transforming growth factor-beta1 and nerve growth factor.


RESULTS
Histological examination of lyophilized and cryopreserved human amniotic membrane showed similar results. Immunohistochemistry showed presence of collagen IV throughout the basement membrane, both in cryopreserved and lyophilized samples. Total protein amount was higher in cryopreserved samples, without statistical significance. Growth factors ELISA did not show statistically significant differences except for fibroblast growth factor basic, with higher levels in cryopreserved amniotic membrane.


CONCLUSIONS
Lyophilization maintains the histological structure of HAM, but seems to cause greater reductions in total protein amount and growth factor concentration than cryopreservation.
Rodríguez-Ares
M Teresa
MT
Department of Ophthalmology, University of Santiago de Compostela (USC) Hospital Complex, Santiago de Compostela, Spain. lopez.mariajesus@gmail.com
López-Valladares
María J
MJ
Touriño
Rosario
R
Vieites
Begoña
B
Gude
Francisco
F
Silva
María T
MT
Couceiro
José
J
D000650:Amnion / Q000378:metabolism* / Q000473:pathology*; D001485:Basement Membrane / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D015925:Cryopreservation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005612:Freeze Drying; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D011506:Proteins / Q000378:metabolism; D014018:Tissue Distribution; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D024141:Collagen Type IV; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D053773:Transforming Growth Factor beta1